1. Field of the Invention
The present invention generally relates to methods of assessing and preventing oxidant stress in vitro and in vivo. More specifically, the present invention relates to methods of measuring nitration and polymerization of proteins which can be used for assessing oxidant stress and methods of preventing nitration and/or polymerization of protein due to oxidant stress.
2. Description of Related Art
It is generally known in the art that superoxide is produced under oxidant stress. This reactive molecule directly or indirectly reacts with macromolecules such as DNAs and proteins and depletes reducing agents such as glutathione in the cells.
Prostaglandin H2 synthases, which are membrane-bound enzymes, catalyze the committed step in the biosynthesis of the prostaglandins and thromboxanes. Prostaglandin H2 synthases have cyclooxygenase and peroxidase activities (1). Prostaglandin H2 synthases add molecular oxygen to arachidonic acid to form prostaglandin G2. Prostaglandin G2 is then rapidly converted to prostaglandin H2 by reduction of the peroxide to a hydroxyl group. Prostaglandin H2 is enzymatically or non-enzymatically converted to prostaglandin F2xcex1, prostaglandin E2, prostaglandin D2, prostaglandin I2 (prostacyclin) or thromboxane A2 (2-4). In addition to constitutively expressed prostaglandin H2 synthase form 1, a second inducible form of prostaglandin H2 synthase, prostaglandin H2 synthase form 2, was discovered in chicken fibroblasts (5) and murine 3T3 cells (6). Prostaglandin H2 synthase form 2 is similar to prostaglandin H2 synthase form 1 with regard to molecular size, subunit composition, and general reaction mechanism. The two prostaglandin H2 synthase isoforms have 60% identity in their primary sequences (7).
Prostaglandin biosynthesis is the target for non-steroidal anti-inflammatory drugs (NSAIDs). Prostaglandin H2 synthase is the primary target of aspirin, indomethacin, ibuprofen, and other NSAIDs (5,8). Increased expression of both inducible nitric oxide (NO) synthase and prostaglandin H2 synthase form 2 has been reported in intestinal inflammation (9-11).
Cytochrome c induces apoptosis by translocation from the mitochondrial membrane to the cytoplasm followed by binding to the apoptotic protease activating factor-1 (Apaf-1) which activates caspases (12). Nitration and/or polymerization of cytochrome c in mitochondria may compromise its translocation and/or its caspase activation function.
Nitration of several amino acid residues of proteins is a result of the reaction of NO with superoxide which is produced under oxidant stress to form peroxynitrite, a potent toxic oxidant. Recently, it was reported that nitrotyrosine was also formed via a tyrosyl radical produced during catalysis of prostaglandin H2 synthase form 2 (13). Nitration of caspase-3 (14), ribonucleotide reductase (15), and cytochrome P450 2B1 (16) resulted in loss of catalytic activity. Interestingly, nitration of cysteine residue of ovine prostaglandin H2 synthase form 1 enhanced catalytic activity by alteration of secondary structure of the enzyme (17).
While it is well known in the art that oxidant stress can lead to further problems within the body which, as set forth above, there are no methods or products which limit or eliminate oxidant stress. These problems can include cancer and other ailments. Accordingly, it is desirable to develop new methods and markers for determining oxidant stress.
According to the present invention, there is provided a method of assessing oxidant stress by measuring polymerization of proteins. Also provided is a marker for oxidant stress which includes a polymerized protein. A kit for use in assessing oxidant stress, the kit including an assay for detecting polymerized proteins is also provided. A method of lowering oxidant stress by administering to a patient an effective amount of at least one reducing agent is also provided. A pharmaceutical composition for lowering oxidant stress, the pharmaceutical having an effective amount of reducing agent and a pharmaceutically acceptable carrier is also provided.